Ligand recognition and binding regulates almost all biological processes, including immune recognition, cell signalling and communication, transcription and translation, intracellular signalling, and enzymatic catalysis. As a result, there is a longstanding interest in the art in identifying molecules which can be used as follows: to serve as agonists or antagonists of ligands such as hormones, growth factors, or neurotransmitters; to induce B-cell (antibody-mediated) or T-cell (cell-mediated) immunity; to induce catalysis of chemical reactions; and to regulate gene expression at the level of transcription or translation. A main reason for this interest is the desire to directly use these biologically active molecules as drugs or, if necessary, to convert these molecules into derivatives which can function as drugs.
Many biological ligands are proteins or peptides. This list includes the majority of hormones, growth factors, neuroactive molecules, and immune epitopes. For this reason, initial efforts to develop agonists or antagonists of receptor- or enzyme-mediated biological activities involved peptide design and synthesis. However, peptides that have been found to possess desirable biological activities are often unsuitable as drugs. To become drugs, the peptides often need to be converted to derivatives or structural analogs, i.e., peptide mimetics, which, unlike most peptides, possess satisfactory pharmacokinetics and stability properties. Many publications describing the development of medicinally useful or promising peptidomimetics have appeared; some recent examples include Rudy Baum, in Chemical & Engineering News, Jan. 18, 1993, page 33; Hirschmann, R. et al. J. Am. Chem. Soc., 1992, 114, 9699-9701; Hirschmann, R. et al. J. Am. Chem. Soc., 1992, 114, 9217-9218.
The discovery of biologically active compounds can be a difficult, time-consuming, and extremely expensive process. A key problem in this area is the identification of a single chemical structure, out of a large number of possible relevant structures, that possesses the desired properties. When the discovery process employs a sequential strategy of structure-design, synthesis, and biological testing, the identification of a desirable chemical structure becomes extremely laborious. To circumvent this highly demanding task, libraries of large numbers of molecules of diverse structures can be prepared. Ideally such libraries can be screened and evaluated rapidly.
Much of the work in this area of library synthesis and screening has been done with peptides, e.g., the approaches of Geysen (Geysen et. al. Molecular Immunology, 1986, 23, 709-715; Geysen et al. J. Immunologic Methods, 1987, 102, 259-274), Fodor (Fodor et al., Science, 1991, 251, 767-773) and Houghten (Houghten et al., Nature, 1991, 354, 84-86). However, such libraries are limited in terms of the number of possible structural variants that can be prepared, tested and identified in a given experiment.
The invention of truly random libraries of polymeric synthetic test compound, in which a single polymeric species arising from a combination of subunits is attached to a single solid support, marked a breakthrough in the discovery of biologically active compounds which are peptides or, very importantly, peptide mimetics (see, U.S. patent application Ser. No. 07/717,454, filed Jun. 19, 1991, entitled "Random Bio-Oligomer Library, a Method of Synthesis Thereof, and a Method of Use Thereof" and U.S. patent application Ser. No. 07/456,845, filed Jul. 2, 1990, entitled "Random Peptide Library, a Method of Synthesis Thereof and a Method of Use Thereof").
Nonpeptidic organic compounds, such as peptide mimetics, can often surpass peptide ligands in affinity for a certain receptor or enzyme. The binding of biotin and avidin, the tightest ever recorded, involves association of a non-peptidic organic structure (biotin) with a protein (avidin). An effective strategy for rapidly identifying high affinity biological ligands, and ultimately new and important drugs, requires rapid construction and screening of diverse libraries of non-peptidic structures containing a variety of structural units capable of establishing one or more types of interactions with a biological acceptor (e.g., a receptor or enzyme), such as hydrogen bonds, salt bridges, .pi.-complexation, hydrophobic effects, etc. However, work on the generation and screening of synthetic test compound libraries containing nonpeptidic molecules is now in its infancy. One example from this area is the work of Ellman and Bunin on a combinatorial synthesis of benzodiazepines on a solid support (J. Am. Chem. Soc. 114, 10997, (1992); see Chemical and Engineering News, Jan. 18, 1993, page 33).
A key unsolved problem in the area of generation and use of nonpeptide libraries is the elucidation of the structure of molecules selected from a library that show promising biological activity.
An attempt to uncover the structures of peptides selected from a library using unique nucleotide sequence codes, which are synthesized in tandem with the peptide library, has recently been described by Brenner and Lerner (Brenner, S. and Lerner, R. A. Proc. Nat'l. Acad. Sci. USA, 1992 89, 5381-5383). The nucleotide sequence of the code attached to each peptide must be amplifiable via the polymerase chain reaction (PCR). However, nucleotide synthesis techniques are not compatible with all of the synthetic techniques required for synthesis of many types of molecular libraries. Furthermore, the close proximity of nucleotide and synthetic test compound in the library, which can result in interactions between these molecules interfering with the binding of the ligand with a target receptor or enzyme during the biological assay, also limits this approach. The nucleotide component of the library can also interfere during biological assays in a variety of other ways.
Kerr et al. (J. Am. Chem. Soc., 1993, 115, 2520-2531) reported synthesizing solution phase libraries of peptides, containing non-natural amino acid residues, in parallel with peptide coding strands. The peptide ligand and its coding strand in this library are covalently joined together, which allows isolation and sequence determination of pairs of synthetic test compound and corresponding code. However, as with the nucleic-acid-encoded library described by Brenner and Lerner, supra, the coding peptide may interfere with the screening assay. Moreover, the requirement for purification of sufficient amounts of material from the library with the affinity selection method, in order to obtain the sequence of the coding peptides, precludes synthesis of libraries of more than a few thousand species.
Thus, there is a need in the art for new, general, and versatile methods for generating and screening libraries of compounds belonging to a variety of chemical classes. There is a further need for effective methods for elucidating the structures of compounds selected from the library as a result of screening, whose structures cannot be determined by traditional techniques, e.g., Edman degradation or mass spectrometry alone. Yet another need in the art is a molecular coding system that will not interact in screening assays or influence the binding of the synthetic test compound through proximity effects.
Citation or identification of any reference herein shall not be construed as an admission that such reference is available as prior art to the present invention.